Bien que l'initiation de la traduction soit un processus conservé entre les bactéries, nous avons montré que le mécanisme par lequel les ARNm structurés sont reconnus et adaptés sur le ribosome diffère chez Staphylococcus aureus, un micro-organisme avec un bas taux de G+C et chez Escherichia coli. Une particularité du ribosome de S. aureus est l'absence de la protéine ribosomale S1, qui non seulement est plus courte que celle de E. coli mais qui possède également une organisation distincte des domaines. Mes expériences suggèrent que la protéine S1 (SauS1) favorise spécifiquement l'initiation de la traduction de l'opéron α-psm 1-4 en liant son ARNm hautement structuré. En outre, il influence aussi l'expression et la production de facteurs de virulence comme les exotoxines (α-haemolysine, δ-hémolysine et γ- hémolysine) et les exoenzymes (protéases et lipases). En plus de son rôle dans la traduction, SauS1 pourrait être impliquée dans d'autres processus cellulaires tels que le métabolisme de l'ARN et la régulation par des ARN non-codants (ARNnc). Elle forme des complexes in vivo avec plusieurs ARNnc dont la stabilité serait affectée dans la souche délétée du gène rpsA codant S1. SauS1 a donc une activité chaperonne favorisant la cinétique d’appariement entre deux molécules d'ARN et au moins dans un cas, elle stimule la reconnaissance entre un ARNnc et son ARN cible. Ainsi, SauS1 appartient à une nouvelle classe de chaperons d'ARN qui jouent un rôle clé dans la régulation du virulon de S. aureus. / Even if translation initiation is a conserved process among bacteria, we have recently shown that low G+C content Gram-positive, such as Staphylococcus aureus, differ from E. coli on the mechanism by which structured mRNAs are recognized and adapted on the ribosome. One peculiarity of the S. aureus ribosome is the absence of ribosomal protein S1, which is shorter than E. coli S1 and has different domains organization. My work could demonstrate that S. aureus S1 (SauS1) specifically promotes translation initiation of the α-psm 1-4 operon by binding its highly structured mRNA. Moreover, it influences the expression and production of other exotoxins (α-haemolysin, δ-haemolysin and γ-haemolysins) and exoenzymes (proteases and lipases). Besides its role in translation, SauS1 could be implicated in other cellular processes such as RNA maturation/degradation and sRNA-mediated regulation. It forms in vivo complexes with several sRNAs whose level is affected in a strain deleted of rpsA gene, coding for S1. Preliminary results show that SauS1 has a chaperone activity promoting the kinetic of annealing of two model RNA molecules and at least in one case, we could demonstrate that it stimulates the recognition between a sRNA and its target RNA. Taken together, SauS1 belongs to a new class of RNA chaperones that play key roles in the regulation of S.aureus virulon.

The waterborne bacterial pathogen Vibrio cholerae uses a process of cell-to-cell communication called quorum sensing (QS) to coordinate transcription of four sRNAs (Qrr1-4; quorum regulatory RNAs) in response to changes in extracellular QS signals that accumulate with cell density. The Qrr sRNAs are predicted to negatively control translation of several mRNAs, including hapR, which encodes the master QS transcription factor that controls genes for virulence factors, biofilm formation, protease production, and DNA uptake. The Qrr sRNAs are also predicted to positively control vca0939, which encodes a GGDEF family protein that promote biofilm formation by elevating intracellular levels of the second messenger molecule c-di-GMP. Using complementary in vivo, in vitro, and bioinformatic approaches, I showed that Qrr sRNAs base-pair with and repress translation of the mRNA encoding HapR. A single nucleotide mutation in Qrr RNA abolishes hapR pairing and thus prevents cholera toxin production and biofilm formation that are important in disease, and also alters expression of competence genes required for uptake of DNA in marine settings. I also demonstrated that base-pairing of the Qrr sRNAs with vca0939 disrupts an inhibitory structure in the 5' UTR of the mRNA. Qrr-activated translation of vca0939 was sufficient to promote synthesis of c-di-GMP and early biofilm formation in a HapR-independent manner. Thus, these studies define the non-coding Qrr sRNAs as a critical component allowing V. cholerae to sense and respond to environmental cues to regulate important developmental processes such as biofilm formation.

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L'identification des interactions survenant au niveau moléculaire joue un rôle crucial pour la compréhension du vivant. L'objectif de ce travail a consisté à développer des méthodes permettant de modéliser et de prédire ces interactions pour le métabolisme et la régulation de la transcription. Nous nous sommes basés pour cela sur la modélisation de ces systèmes sous la forme de graphes et d'automates. Nous avons dans un premier temps développé une méthode permettant de tester et de prédire la distribution du flux au sein d'un réseau métabolique en permettant la formulation d'une à plusieurs contraintes. Nous montrons que la prise en compte des données biologiques par cette méthode permet de mieux reproduire certains phénotypes observés in vivo pour notre modèle d'étude du métabolisme énergétique du parasite Trypanosoma brucei. Les résultats obtenus ont ainsi permis de fournir des éléments d'explication pour comprendre la flexibilité du flux de ce métabolisme, qui étaient cohérentes avec les données expérimentales. Dans un second temps, nous nous sommes intéressés à une catégorie particulière d'ARN non codants appelés sRNAs, qui sont impliqués dans la régulation de la réponse cellulaire aux variations environnementales. Nous avons développé une approche permettant de mieux prédire les interactions qu'ils effectuent avec d'autres ARN en nous basant sur une prédiction des interactions, une analyse par enrichissement du contexte biologique de ces cibles, et en développant un système de visualisation spécialement adapté à la manipulation de ces données. Nous avons appliqué notre méthode pour l'étude des sRNAs de la bactérie Escherichia coli. Les prédictions réalisées sont apparues être en accord avec les données expérimentales disponibles, et ont permis de proposer plusieurs nouvelles cibles candidates. / The identification of the interactions occurring at the molecular level is crucial to understand the life process. The aim of this work was to develop methods to model and to predict these interactions for the metabolism and the regulation of transcription. We modeled these systems by graphs and automata.Firstly, we developed a method to test and to predict the flux distribution in a metabolic network, which consider the formulation of several constraints. We showed that this method can better mimic the in vivo phenotype of the energy metabolism of the parasite Trypanosoma brucei. The results enabled to provide a good explanation of the metabolic flux flexibility, which were consistent with the experimental data. Secondly, we have considered a particular class of non-coding RNAs called sRNAs, which are involved in the regulation of the cellular response to environmental changes. We developed an approach to better predict their interactions with other RNAs based on the interaction prediction, an enrichment analysis, and by developing a visualization system adapted to the manipulation of these data. We applied our method to the study of the sRNAs interactions within the bacteria Escherichia coli. The predictions were in agreement with the available experimental data, and helped to propose several new target candidates.

Cells sense the properties of the surrounding environment and convert this information into changes in gene expression. Bacteria are, in contrast to many multi-cellular eukaryotes, remarkable in their ability to cope with rapid environmental changes and to endure harsh and extreme milieus. Previously, control of gene expression was thought to be carried out exclusively by proteins. However, it is now clear that small regulatory RNAs (sRNA) also carry out gene regulatory functions. Bacteria such as E. coli harbor a large class of sRNAs that bind to mRNAs to alter translation and/or mRNA stability. By identifying mRNAs that are targeted by sRNAs, my studies have broadened the understanding of the mechanisms that underlie sRNA-dependent gene regulation, and have shed light on the impact that this type of regulation has on bacterial physiology. Control of gene expression often relies on the interplay of many regulators. This interplay is exemplified by our discovery of mutual regulation between the sRNA MicF and the globally acting transcription factor Lrp. Through double negative feedback, these two regulators respond to nutrient availability in the environment which results in reprogramming of downstream gene expression. We have also shown that both the transcription factor CsgD, and the anti-sigma factor FlgM, are repressed by the two sRNAs OmrA and OmrB, suggesting that these sRNAs are important players in the complex regulation that allow bacteria to switch between motility and sessility. Bacterial populations of genetically identical individuals show phenotypic variations when switching to the sessile state due to bistability in gene expression. While bistability has previously been demonstrated to arise from stochastic fluctuations in transcription, our results suggest that bistability possibly may arise from sRNA-dependent regulatory events also on the post-transcriptional level.

Bacteria are exposed to various environmental conditions during their growth. They have to cope with rapid changes in temperature, lack of nutrition, etc. To survive, bacteria alter their gene expression. One type of regulation of gene expression is regulation by small RNAs (sRNAs). In bacteria, a well-studied sRNA is 6S RNA that binds to the RNA polymerase holoenzyme. However, 6S RNA has not been identified in several bacterial species. Mycobacteria are a genus that probably does not have 6S RNA. Instead, Mycobacterium smegmatis possess another sRNA - Ms1. Ms1 structurally resembles 6S RNA and indeed it was first identified as a 6S RNA structural homologue. However, Ms1 binds to RNAP devoid of any sigma factor, and, therefore, is significantly distinct from 6S RNA. This work describes regulation of expression of Ms1. DNA fragments of different length from the region upstream of the Ms1 gene were prepared. These fragments were fused to the lacZ reporter gene and their activity was tested in different growth phases and under stress. This allowed identification and characterization of the core promoter sequence and regulatory sequences that might interact with transcription factor(s). Promoter activity increased with increased length of the promoter fragment and after transition into stationary...

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